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Parasitic interactions between Claviceps purpurea strains in wheat and an acute necrotic host response
Mycol. Res. 95 (7): 807-810 (1991)
Printed
in
Great Britain
807
Parasitic interactions between Claviceps purpurea strains in wheat and an acute necrotic host response
D.JANET SWAN AND PETER G.MANTLE Department of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2A Y, u.K.
Concurrent inoculation of wheat florets with pairs of distinctive strains of Claviceps purpurea gave ergot sclerotia in which both strains co-existed as indicated by their alkaloid content, the ability to produce the sexual stage and sclerotial morphology. Production of heterogeneous sclerotia may facilitate hybridization of strains during subsequent ascogeny. A strain notable for its parasitic vigour and failure to biosynthesize ergot alkaloids displaced another strain when the second inoculum was introduced around infected ovaries 7 days after initial inoculation. The necrosis of affected spikelets which occurred in response to the interparasitic competition is recognized as a novel phenomenon in ergot-host relationships. The diverse biological forms of ergot fungi encompassed, even in the U.K., by Claviceps purpurea (Fr.) Tul. are partly expressed in parasitism in the extent to which they are restricted to certain groups of host grasses (Loveless, 1971). There may also be characteristic differences in sclerotial and conidial shape as well as in the pattern of indole alkaloids which are elaborated within the sclerotia (Mantle, Shaw & Doling, 1977). Several forms may exist within a locality so that, for example, wheat crops may be exposed concurrently to inoculum of more than one biological form of ergot pathogen. The extent to which more than one biotype may contribute in a mixed inoculum to the parasitic destruction of a wheat ovary has apparently not been studied, nor has the extent to which more than one biotype can, by concerted proliferation, give rise to a sclerotium of heterogeneous composition. Neither of these topics is likely to have significant direct impact on ergot pathology since conidial inoculum usually consists of several or many spores transmitted from honeydew by rainsplash, head-to-head contact or by insect vectors. When, as a result of infection by C. purpurea, an ovary ceases to be a fertilizable gynoecium with potential for seed development the extent or type of sclerotial proliferation which follows is of little consequence to the grain yield and quality of that crop. However, there are much wider biological consequences if initiation of an ascogonium and antheridium involved in perithecial formation is from adjacent hyphae in a genetically heterogeneous sclerotium composed of an intimate mixture of two genotypes. The prospect of genetic mixing would provide the potential for exploitable variety which otherwise seems to be precluded in the cryptic formation of stromatal initials. It would, of course, require that mating antheridium and ascogonium do not have to arise from a single hypha as is depicted for some free-living homothallic ascomycetes in which sexual reproduction is initiated in a more open mycelial matrix. The present study of interactions between distinctive forms
of C. purpurea during parasitism has not only confirmed the principle of co-existence but has also revealed an unexpected novel response in the host.
EXPERIMENT AL Experiment 1 Four days prior to anthesis twenty groups of ten uniformly sized ears of field-grown winter wheat (cv. Maris Huntsman) were selected. Four strains (A-D) of C. purpurea were chosen for their distinctive ergot alkaloid content (Table 1) and differing morphological features. All had been isolated from sclerotia parasitizing U.K. cereals; strain B is strain 12/2 of Corbett, Richardson & Mantle, (1974). Spore suspensions of each strain (2 X 10 7 and 4 X 10 4 spores/ml) were made by diluting honeydew (stored under liquid nitrogen) with distilled water. Spore suspensions were inoculated alone, or in combinations of equal volumes, into 75 % of spikelets in each ear of a group of plants. Protected from birds, the ears were grown to maturity and threshed by hand. Ergots were coded according to the inoculum used (e.g. Ab used inoculum containing 10 7 spores of strain A ml- 1 and 2 X 10 4 spores of strain B ml- 1 ). Three months after harvest one hundred ergots taken at random from each group were soaked in distilled water for twenty-four hours and placed 2-4 mm beneath the surface of moist vermiculite and incubated at 4 °C for seven weeks. Further incubation was at room temperature (ca 20°) until no further stromata arose. From the onset of stroma production the number of stromata in each group was scored periodically. The total alkaloid content of a 1 g sample of ergots, chosen to reflect the range of size produced, was determined in each group (Mantle et al., 1977). The individual alkaloids were resolved by silica gel tic of extracts in ethyl acetate/ ethanoljdimethyl formamide (19:0'1:1'9 or 26:0'1:1'9 according, respectively, to whether the ergotamine
Parasitic interactions in wheat
808
control (uninoculated) ears, using a seedling emergence test. The seeds were sown in an open plot, protected with net, at the Chelsea Physic Garden, London, during November, and the number of emerged seedlings was recorded nine weeks later. The alkaloid content of individual, or representative groups of, ergot sclerotia was analysed as in Experiment I to identify any co-existence of strains. Also the extent of strain mixing within sclerotia was measured by alkaloid analysis of the younger (proximal) third of separate sclerotia.
group / ergotoxine group ratio was adequately diagnostic or whether the components of the ergotoxine group needed to be resolved). Twelve representative ergots were selected from strain combinations AB and AD, and three of each of the single strains A, Band D. Alkaloids were extracted from each individual sclerotium and resolved by tic as above.
Experiment 2 Groups of uniformly sized ears of winter wheat (cv. Maris Huntsman) were inoculated four days prior to anthesis. The inoculum contained honeydew-derived spores (2 x 10 7 ml- 1) and consisted of strains Band E of C. purpurea alone or in equal combination (Table 2). Strain B was alkaloid-free and produced short broad sclerotia. Strain E was used on account of having characteristically long sclerotia containing abundant alkaloid and was strain 29/4 of Corbett et al. (1974). Five central spikelets on one side of each ear were inoculated. A group of ears inoculated with strain B or E was inoculated again, seven days later, with the other strain. (The pathogenicity of this later inoculum was confirmed by injection into pre-anthesis ears in the same plot). In the second inoculations care was taken to superimpose the strains by inoculating through the original needle wounds. The plants were grown to maturity and harvested by hand. Ears from each of the treatment groups were matched for size, to improve comparability, and the number and weight of ergots and seeds was recorded for each ear. The quality of the seeds from ears inoculated with strain E only and with strain E followed by strain B was compared with that of seeds from
RESULTS Experiment 1 Nine days after inoculation honeydew exudation was prolific in all wheat ears except whenever strain B had been inoculated as the sole, equal or dominant component (groups B, b, AB and aB). All the mature sclerotia were of similar and typical C. purpurea shape except those in groups B, band aB which were considerably shorter and broader. The results of germination tests for the sclerotia derived from a single strain show a clear division into two groups: strains C and A had a germination rate of 30-35 % while in strains Band D the rate was only 1-2 % (Table I). All the combinations containing strain B also showed light contamination with zygomycete hyphae which were apparent ten weeks after incubation commenced. Strains with poor germination generally exerted this dominant influence when occurring as part of a mixture with a strain which alone germinated quite readily. Unequal mixtures of inoculum generally gave rise to
Table 1. Germination and alkaloid content of ergot sclerotia arising from mixed and single strain inoculations of wheat, as indicators of the heterogeneity of parasitic development from certain combinations of strains of C. purpurea Ergot strains
alone or combined' A a B b C D d AB Ab aB AC Ac aC AD Ad aD CD Cd cD
Stromata from 100 ergots 61 61 2 1 52 52 2 I
4 5 16 46 99 19
a 6 4 4 49 7
Sclerotia with 0-6 stromata
a
2
69 61
19 24
98 99 70 71
2 1 17 13
99 99 97 97 94
3 7
3
4
5
5 4
2
3 1
I
8 10 1
3 5
(%)
(%)
Dominant strain according to alkaloids·t
31} 39
0'26
A
a
B
0'41
C
0'40
D
3
0'17
3 6 27
0'26 0'03 0'37
56 14
0'29 0'35
0 4
0'36 0'32
2 2 29
0'39 0'39 0'51 0'29
A A B A+C A C A+D A D C+D C C+?D
3D} 29
a
1 2
I
3
73 44
13 30
II
2 5
3
86 100
10
16 2
I
1
96 98
2
2
98 71 95
1 17
I
2
3
Sclerotial alkaloid
~}
I
2
6
Sclerotial germination
I
7 2
3
I
2
5
, Capital. high spore inoculum; lower case, low spore inoculum. t Strain A: ergotoxine > ergotamine; ergosine: ergocornine and ergokryptine > ergocristine. Strain B; none. Strain C: ergotoxine < ergotamine; ergosine. Strain D: ergotoxine = ergotamine; ergosine, ergocornine and ergokryptine < ergocristine.
809
D. Janet Swan and Peter G. Mantle sclerotia of the type dominating the inoculum. For groups of sclerotia derived from inoculation with an approximately equal number of viable conidia of two distinct strains of C. purpurea the combined evidence of qualitative and quantitative alkaloid content, and that of elaboration of the sexual stage, consistently indicated heterogeneity (Table 1). That the heterogeneity was at least principally intra-sclerotia!, and not just the result of homogenizing a mixed population, was demonstrated by the results of analysis of individual sclerotia of the AB and AD combinations and of strains A. B and D alone. The three sclerotia of strain B were all alkaloid-free. All twelve sclerotia from the AB group had the alkaloid spectrum characteristic of strain A. Of the twelve sclerotia from the AD group, one had ergocristine but no ergocornine or ergokryptine (as those of strain D) and another had ergocornine and/or ergokryptine, strongly indicating the presence of both strains A and D.
Where strain B was inoculated first (expected to yield about 3 sclerotia in each ear) the addition of strain E, one week later, reduced by half the number of ergots (Table 2). Alkaloid analysis of individual sclerotia showed that fifteen had none and so were considered to be pure strain B. One sclerotium contained 0'39% alkaloid which at least indicates a predominance of strain E. Three sclerotia had low alkaloid contents (0'01-0'08%) indicating the presence of strain E but suggesting that strain B may also have been present. It is concluded that strain E reduced sclerotia production by the already established strain B by approximately 50%. However, there is no evidence that it could supplant strain Band produce a strain E sclerotium in a floret either where strain B would have yielded a sclerotium or where strain B would have aborted. The one ergot which appeared to be typical strain E may have developed in a previously uninoculated terminal floret of a spikelet. However, when inoculation with strain E was followed one week later by the addition of strain B the majority of infections aborted at the sphacelial stage. Instead of about 12 ergots developing in each ear, as was recorded for strain E alone, each double inoculated ear yielded, on average, less than one sclerotium (Table 2). One to two weeks after the superimposition of strain B death of the floral pads of the inoculated spikelets was evident. Necrosis was confined to inoculated spikelets and these later became colonized by saprotrophic fungi. The high alkaloid content (0'43-0'66 %) of all seven surviving sclerotia in this group indicates that they were pure strain E since individual analysis of seven other pure strain E ergots showed an alkaloid range of 0'22-0'52 %. This suggests that they may have developed in terminal florets to which strain B had not been introduced. Apparently the application of strain B spores around an ovary with established strain E infection completely eliminated sclerotium production by the resident pathogen. It is not clear whether this was an interaction solely at the fungal level or whether it was caused by nutrient deprivation resulting from the dramatic necrosis of the spikelets directly involved in the experiment. Irrespective of the mechanisms of abortion of strain E infection, it is notable that strain B was unable to become an established parasite and form sclerotia. The mean seed weight was remarkably constant between treatments and was not affected by the presence of ergots (Table 2). Similarly, percentage seedling emergence was not markedly affected by ergot infection.
Experiment 2 Ears inoculated with strain B or strain E alone yielded ergots with no alkaloid or 0'31 % alkaloid, respectively, and which differed not only in their abundance but also in their morphology. Strain B honeydew exudate was sparse and the pathogen yielded fewer sclerotia although it was almost as efficient as strain E in reducing seed yield (Table 2). Inoculation with the strain mixture BE yielded ergots which, in abundance and alkaloid content (0'15 %), were intermediate between those of strain B or E alone. This is consistent with the results of experiment 1. Analysis of the younger part of other sclerotia from a mixed inoculum showed that not only was alkaloid present in each sclerotium (mean 0'14%; range 0'10-0'18%) but its production had persisted throughout parasitism. The proximal portion of pure strain E sclerotia contained a mean alkaloid content of 0'36% (range 0'20-0'55 %). The lower alkaloid content of the ergots from mixed inoculum is consistent with the presence of the non-alkaloid producing strain B. Parasitism of host tissues by strain B is characterized by development of more sphacelial tissue and larger mature ergots than are produced by strain E. The results suggest that, in spite of this difference in sphacelial and sclerotial growth form, when strains Band E were simultaneously inoculated into a floret they both contributed to the growing sclerotium during most, if not all, of its development.
Table 2. Comparison of seed and ergot production in wheat ears inoculated with C. purpurea Ergot strains alone. together ( + ) or in sequence (-+) at a 7-day interval
Wheat ears (no.) Mean seeds/ear Mean seed wt (mg) Mean ergots/ear Mean ergot wt (mg) Seedling emergence (%t • From matched groups of 16 ears.
Control
B
E
B+E
B-+E
E-+B
80 47'4 54'5
31 34'5 54'6 2'6 37'1
123 27'7 54'0 14'6 40'5 94'5'
31 29'4 59'2 8'9 28'5
11
34'8 56'2 1'5 84'5
16 31'4 55'0 0'5 77'7 83'0'
88'8'
810
Parasitic interactions in wheat
DISCUSSION It is clear that mixed populations of C. purpurea conidia may give rise to a sphacelial fructification consisting of the components of the mixture, even in a disparate ratio of 500 : I, co-existing then to form a heterogeneous sclerotium from which the extent of differentiation of a sexual stage may even be dominated by a strain which is shy to produce stromata. The extent to which intimate admixture of two or more characteristic strains could hybridize in stromata elaborated from heterogeneous sclerotia deserves experimental study. It is notable also that the co-habitational tolerances demonstrated contrast with the necrotic effect of introducing one pathogen, (Received for publication 5 July 1990)
strain B, to an established infection of another. This remarkable host response was sustained in repeated experiments in two subsequent years, confirming the phenomenon and raising questions concerning the mechanism of the interactions.
REFERENCES Corbett. K.. Dickerson, A. G. & Mantle. P. G. (1974). Metabolic studies on Claviceps purpurea during parasitic development on rye. Journal of General Microbiology 84, 39-58. Loveless, A. R. (I971). Conidial evidence for host restriction in Claviceps purpurea. Transactions of the British Mycological Society 56, 419-434. Mantle, P. G., Shaw, S. & Doling, D. A. (1977). Role of weed grasses in the etiology of ergot disease in wheat. Annals of Applied Biology 86, 339-351.
Printed
in
Great Britain
807
Parasitic interactions between Claviceps purpurea strains in wheat and an acute necrotic host response
D.JANET SWAN AND PETER G.MANTLE Department of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2A Y, u.K.
Concurrent inoculation of wheat florets with pairs of distinctive strains of Claviceps purpurea gave ergot sclerotia in which both strains co-existed as indicated by their alkaloid content, the ability to produce the sexual stage and sclerotial morphology. Production of heterogeneous sclerotia may facilitate hybridization of strains during subsequent ascogeny. A strain notable for its parasitic vigour and failure to biosynthesize ergot alkaloids displaced another strain when the second inoculum was introduced around infected ovaries 7 days after initial inoculation. The necrosis of affected spikelets which occurred in response to the interparasitic competition is recognized as a novel phenomenon in ergot-host relationships. The diverse biological forms of ergot fungi encompassed, even in the U.K., by Claviceps purpurea (Fr.) Tul. are partly expressed in parasitism in the extent to which they are restricted to certain groups of host grasses (Loveless, 1971). There may also be characteristic differences in sclerotial and conidial shape as well as in the pattern of indole alkaloids which are elaborated within the sclerotia (Mantle, Shaw & Doling, 1977). Several forms may exist within a locality so that, for example, wheat crops may be exposed concurrently to inoculum of more than one biological form of ergot pathogen. The extent to which more than one biotype may contribute in a mixed inoculum to the parasitic destruction of a wheat ovary has apparently not been studied, nor has the extent to which more than one biotype can, by concerted proliferation, give rise to a sclerotium of heterogeneous composition. Neither of these topics is likely to have significant direct impact on ergot pathology since conidial inoculum usually consists of several or many spores transmitted from honeydew by rainsplash, head-to-head contact or by insect vectors. When, as a result of infection by C. purpurea, an ovary ceases to be a fertilizable gynoecium with potential for seed development the extent or type of sclerotial proliferation which follows is of little consequence to the grain yield and quality of that crop. However, there are much wider biological consequences if initiation of an ascogonium and antheridium involved in perithecial formation is from adjacent hyphae in a genetically heterogeneous sclerotium composed of an intimate mixture of two genotypes. The prospect of genetic mixing would provide the potential for exploitable variety which otherwise seems to be precluded in the cryptic formation of stromatal initials. It would, of course, require that mating antheridium and ascogonium do not have to arise from a single hypha as is depicted for some free-living homothallic ascomycetes in which sexual reproduction is initiated in a more open mycelial matrix. The present study of interactions between distinctive forms
of C. purpurea during parasitism has not only confirmed the principle of co-existence but has also revealed an unexpected novel response in the host.
EXPERIMENT AL Experiment 1 Four days prior to anthesis twenty groups of ten uniformly sized ears of field-grown winter wheat (cv. Maris Huntsman) were selected. Four strains (A-D) of C. purpurea were chosen for their distinctive ergot alkaloid content (Table 1) and differing morphological features. All had been isolated from sclerotia parasitizing U.K. cereals; strain B is strain 12/2 of Corbett, Richardson & Mantle, (1974). Spore suspensions of each strain (2 X 10 7 and 4 X 10 4 spores/ml) were made by diluting honeydew (stored under liquid nitrogen) with distilled water. Spore suspensions were inoculated alone, or in combinations of equal volumes, into 75 % of spikelets in each ear of a group of plants. Protected from birds, the ears were grown to maturity and threshed by hand. Ergots were coded according to the inoculum used (e.g. Ab used inoculum containing 10 7 spores of strain A ml- 1 and 2 X 10 4 spores of strain B ml- 1 ). Three months after harvest one hundred ergots taken at random from each group were soaked in distilled water for twenty-four hours and placed 2-4 mm beneath the surface of moist vermiculite and incubated at 4 °C for seven weeks. Further incubation was at room temperature (ca 20°) until no further stromata arose. From the onset of stroma production the number of stromata in each group was scored periodically. The total alkaloid content of a 1 g sample of ergots, chosen to reflect the range of size produced, was determined in each group (Mantle et al., 1977). The individual alkaloids were resolved by silica gel tic of extracts in ethyl acetate/ ethanoljdimethyl formamide (19:0'1:1'9 or 26:0'1:1'9 according, respectively, to whether the ergotamine
Parasitic interactions in wheat
808
control (uninoculated) ears, using a seedling emergence test. The seeds were sown in an open plot, protected with net, at the Chelsea Physic Garden, London, during November, and the number of emerged seedlings was recorded nine weeks later. The alkaloid content of individual, or representative groups of, ergot sclerotia was analysed as in Experiment I to identify any co-existence of strains. Also the extent of strain mixing within sclerotia was measured by alkaloid analysis of the younger (proximal) third of separate sclerotia.
group / ergotoxine group ratio was adequately diagnostic or whether the components of the ergotoxine group needed to be resolved). Twelve representative ergots were selected from strain combinations AB and AD, and three of each of the single strains A, Band D. Alkaloids were extracted from each individual sclerotium and resolved by tic as above.
Experiment 2 Groups of uniformly sized ears of winter wheat (cv. Maris Huntsman) were inoculated four days prior to anthesis. The inoculum contained honeydew-derived spores (2 x 10 7 ml- 1) and consisted of strains Band E of C. purpurea alone or in equal combination (Table 2). Strain B was alkaloid-free and produced short broad sclerotia. Strain E was used on account of having characteristically long sclerotia containing abundant alkaloid and was strain 29/4 of Corbett et al. (1974). Five central spikelets on one side of each ear were inoculated. A group of ears inoculated with strain B or E was inoculated again, seven days later, with the other strain. (The pathogenicity of this later inoculum was confirmed by injection into pre-anthesis ears in the same plot). In the second inoculations care was taken to superimpose the strains by inoculating through the original needle wounds. The plants were grown to maturity and harvested by hand. Ears from each of the treatment groups were matched for size, to improve comparability, and the number and weight of ergots and seeds was recorded for each ear. The quality of the seeds from ears inoculated with strain E only and with strain E followed by strain B was compared with that of seeds from
RESULTS Experiment 1 Nine days after inoculation honeydew exudation was prolific in all wheat ears except whenever strain B had been inoculated as the sole, equal or dominant component (groups B, b, AB and aB). All the mature sclerotia were of similar and typical C. purpurea shape except those in groups B, band aB which were considerably shorter and broader. The results of germination tests for the sclerotia derived from a single strain show a clear division into two groups: strains C and A had a germination rate of 30-35 % while in strains Band D the rate was only 1-2 % (Table I). All the combinations containing strain B also showed light contamination with zygomycete hyphae which were apparent ten weeks after incubation commenced. Strains with poor germination generally exerted this dominant influence when occurring as part of a mixture with a strain which alone germinated quite readily. Unequal mixtures of inoculum generally gave rise to
Table 1. Germination and alkaloid content of ergot sclerotia arising from mixed and single strain inoculations of wheat, as indicators of the heterogeneity of parasitic development from certain combinations of strains of C. purpurea Ergot strains
alone or combined' A a B b C D d AB Ab aB AC Ac aC AD Ad aD CD Cd cD
Stromata from 100 ergots 61 61 2 1 52 52 2 I
4 5 16 46 99 19
a 6 4 4 49 7
Sclerotia with 0-6 stromata
a
2
69 61
19 24
98 99 70 71
2 1 17 13
99 99 97 97 94
3 7
3
4
5
5 4
2
3 1
I
8 10 1
3 5
(%)
(%)
Dominant strain according to alkaloids·t
31} 39
0'26
A
a
B
0'41
C
0'40
D
3
0'17
3 6 27
0'26 0'03 0'37
56 14
0'29 0'35
0 4
0'36 0'32
2 2 29
0'39 0'39 0'51 0'29
A A B A+C A C A+D A D C+D C C+?D
3D} 29
a
1 2
I
3
73 44
13 30
II
2 5
3
86 100
10
16 2
I
1
96 98
2
2
98 71 95
1 17
I
2
3
Sclerotial alkaloid
~}
I
2
6
Sclerotial germination
I
7 2
3
I
2
5
, Capital. high spore inoculum; lower case, low spore inoculum. t Strain A: ergotoxine > ergotamine; ergosine: ergocornine and ergokryptine > ergocristine. Strain B; none. Strain C: ergotoxine < ergotamine; ergosine. Strain D: ergotoxine = ergotamine; ergosine, ergocornine and ergokryptine < ergocristine.
809
D. Janet Swan and Peter G. Mantle sclerotia of the type dominating the inoculum. For groups of sclerotia derived from inoculation with an approximately equal number of viable conidia of two distinct strains of C. purpurea the combined evidence of qualitative and quantitative alkaloid content, and that of elaboration of the sexual stage, consistently indicated heterogeneity (Table 1). That the heterogeneity was at least principally intra-sclerotia!, and not just the result of homogenizing a mixed population, was demonstrated by the results of analysis of individual sclerotia of the AB and AD combinations and of strains A. B and D alone. The three sclerotia of strain B were all alkaloid-free. All twelve sclerotia from the AB group had the alkaloid spectrum characteristic of strain A. Of the twelve sclerotia from the AD group, one had ergocristine but no ergocornine or ergokryptine (as those of strain D) and another had ergocornine and/or ergokryptine, strongly indicating the presence of both strains A and D.
Where strain B was inoculated first (expected to yield about 3 sclerotia in each ear) the addition of strain E, one week later, reduced by half the number of ergots (Table 2). Alkaloid analysis of individual sclerotia showed that fifteen had none and so were considered to be pure strain B. One sclerotium contained 0'39% alkaloid which at least indicates a predominance of strain E. Three sclerotia had low alkaloid contents (0'01-0'08%) indicating the presence of strain E but suggesting that strain B may also have been present. It is concluded that strain E reduced sclerotia production by the already established strain B by approximately 50%. However, there is no evidence that it could supplant strain Band produce a strain E sclerotium in a floret either where strain B would have yielded a sclerotium or where strain B would have aborted. The one ergot which appeared to be typical strain E may have developed in a previously uninoculated terminal floret of a spikelet. However, when inoculation with strain E was followed one week later by the addition of strain B the majority of infections aborted at the sphacelial stage. Instead of about 12 ergots developing in each ear, as was recorded for strain E alone, each double inoculated ear yielded, on average, less than one sclerotium (Table 2). One to two weeks after the superimposition of strain B death of the floral pads of the inoculated spikelets was evident. Necrosis was confined to inoculated spikelets and these later became colonized by saprotrophic fungi. The high alkaloid content (0'43-0'66 %) of all seven surviving sclerotia in this group indicates that they were pure strain E since individual analysis of seven other pure strain E ergots showed an alkaloid range of 0'22-0'52 %. This suggests that they may have developed in terminal florets to which strain B had not been introduced. Apparently the application of strain B spores around an ovary with established strain E infection completely eliminated sclerotium production by the resident pathogen. It is not clear whether this was an interaction solely at the fungal level or whether it was caused by nutrient deprivation resulting from the dramatic necrosis of the spikelets directly involved in the experiment. Irrespective of the mechanisms of abortion of strain E infection, it is notable that strain B was unable to become an established parasite and form sclerotia. The mean seed weight was remarkably constant between treatments and was not affected by the presence of ergots (Table 2). Similarly, percentage seedling emergence was not markedly affected by ergot infection.
Experiment 2 Ears inoculated with strain B or strain E alone yielded ergots with no alkaloid or 0'31 % alkaloid, respectively, and which differed not only in their abundance but also in their morphology. Strain B honeydew exudate was sparse and the pathogen yielded fewer sclerotia although it was almost as efficient as strain E in reducing seed yield (Table 2). Inoculation with the strain mixture BE yielded ergots which, in abundance and alkaloid content (0'15 %), were intermediate between those of strain B or E alone. This is consistent with the results of experiment 1. Analysis of the younger part of other sclerotia from a mixed inoculum showed that not only was alkaloid present in each sclerotium (mean 0'14%; range 0'10-0'18%) but its production had persisted throughout parasitism. The proximal portion of pure strain E sclerotia contained a mean alkaloid content of 0'36% (range 0'20-0'55 %). The lower alkaloid content of the ergots from mixed inoculum is consistent with the presence of the non-alkaloid producing strain B. Parasitism of host tissues by strain B is characterized by development of more sphacelial tissue and larger mature ergots than are produced by strain E. The results suggest that, in spite of this difference in sphacelial and sclerotial growth form, when strains Band E were simultaneously inoculated into a floret they both contributed to the growing sclerotium during most, if not all, of its development.
Table 2. Comparison of seed and ergot production in wheat ears inoculated with C. purpurea Ergot strains alone. together ( + ) or in sequence (-+) at a 7-day interval
Wheat ears (no.) Mean seeds/ear Mean seed wt (mg) Mean ergots/ear Mean ergot wt (mg) Seedling emergence (%t • From matched groups of 16 ears.
Control
B
E
B+E
B-+E
E-+B
80 47'4 54'5
31 34'5 54'6 2'6 37'1
123 27'7 54'0 14'6 40'5 94'5'
31 29'4 59'2 8'9 28'5
11
34'8 56'2 1'5 84'5
16 31'4 55'0 0'5 77'7 83'0'
88'8'
810
Parasitic interactions in wheat
DISCUSSION It is clear that mixed populations of C. purpurea conidia may give rise to a sphacelial fructification consisting of the components of the mixture, even in a disparate ratio of 500 : I, co-existing then to form a heterogeneous sclerotium from which the extent of differentiation of a sexual stage may even be dominated by a strain which is shy to produce stromata. The extent to which intimate admixture of two or more characteristic strains could hybridize in stromata elaborated from heterogeneous sclerotia deserves experimental study. It is notable also that the co-habitational tolerances demonstrated contrast with the necrotic effect of introducing one pathogen, (Received for publication 5 July 1990)
strain B, to an established infection of another. This remarkable host response was sustained in repeated experiments in two subsequent years, confirming the phenomenon and raising questions concerning the mechanism of the interactions.
REFERENCES Corbett. K.. Dickerson, A. G. & Mantle. P. G. (1974). Metabolic studies on Claviceps purpurea during parasitic development on rye. Journal of General Microbiology 84, 39-58. Loveless, A. R. (I971). Conidial evidence for host restriction in Claviceps purpurea. Transactions of the British Mycological Society 56, 419-434. Mantle, P. G., Shaw, S. & Doling, D. A. (1977). Role of weed grasses in the etiology of ergot disease in wheat. Annals of Applied Biology 86, 339-351.